Mass spectrometry assay method for detection and quantitation of organic acid metabolites

Abstract

A method for determining in a sample, by mass spectrometry, the presence, absence, or amount of one or more analytes is described herein. The run time is less than six minutes. The method includes subjecting the sample to an ionization source under conditions suitable to produce one or more ions detectable by mass spectrometry from each of the one or more analytes, wherein the one or more analytes are derivatized prior to ionization; measuring, in a single injection, by mass spectrometry, the amount of the one or more ions from each of the one or more analytes; and using the measured amount of the one or more ions to determine the amount of each of the one or more analytes in the sample.

What is claimed: 
     
       1. A method for determining in a sample, by mass spectrometry, the presence, absence, or amount of two or more analytes selected from the group consisting of Acetic acid (C2), Propionic acid (C3), Butyric acid (C4), Isobutyric acid (C4), 2-Methyl-butyric acid (C5), Isovaleric acid (C5), Valeric acid (C5), Caproic acid (Hexanoic acid, C6), 3-Methylvaleric acid, 4-Methylvaleric acid (Isocaproic acid), Lactic acid, Pyruvic acid, Fumaric acid, Succinic acid, Malic acid, alpha-Ketoglutaric acid, Aconitic acid, Citric acid, Isocitric acid, and combinations thereof, wherein one of the two or more analytes is selected from the group consisting of: Acetic acid (C2), Propionic acid (C3), Butyric acid (C4), Isobutyric acid (C4), 2-Methyl-butyric acid (C5), Isovaleric acid (C5), Valeric acid (C5), Caproic acid (Hexanoic acid, C6), 3-Methylvaleric acid, 4-Methylvaleric acid (Isocaproic acid); and a different one of the two or more analytes is selected from the group consisting of Lactic acid, Pyruvic acid, Fumaric acid, Succinic acid, Malic acid, alpha-Ketoglutaric acid, Aconitic acid, Citric acid, Isocitric acid, wherein the run time is less than six minutes, the method comprising:
 a) subjecting the sample to an ionization source under conditions suitable to produce one or more ions detectable by mass spectrometry from each of the one or more analytes, wherein the one or more analytes are derivatized prior to ionization; 
 b) measuring, in a single injection, by mass spectrometry, the amount of the one or more ions from each of the one or more analytes; and 
 c) using the measured amount of the one or more ions to determine the amount of each of the one or more analytes in the sample. 
 
     
     
       2. The method of  claim 1 , wherein pivalic acid interference is eliminated. 
     
     
       3. The method of  claim 1 , wherein the sample is derivatized using at least 2,4-Difluorophenyl Hydrazine Hydrochloride or 3-Nitrophenylhydrazine Hydrochloride. 
     
     
       4. The method of  claim 1 , wherein the mass spectrometer is operated in negative mode. 
     
     
       5. The method of  claim 1 , wherein the sample has been purified by liquid chromatography prior to being subjected to an ionization source. 
     
     
       6. The method of  claim 1 , wherein the one or more ions used to determine the amount of each of the one or more analytes are one or more ions selected from the ions in Tables 4 and 5. 
     
     
       7. The method of  claim 1 , wherein the amount of Acetic acid (C2), Propionic acid (C3), and Butyric acid (C4) is determined. 
     
     
       8. The method of  claim 1 , wherein the amount Acetic acid (C2), Propionic acid (C3), Butyric acid (C4) and lactic acid is determined. 
     
     
       9. The method of  claim 1 , wherein the amount Acetic acid (C2), Propionic acid (C3), Isobutyric acid (C4), and Butyric acid (C4) is determined. 
     
     
       10. The method of  claim 1 , wherein the amount Acetic acid (C2), Propionic acid (C3), Isobutyric acid (C4), Butyric acid (C4), 2-Methyl-butyric acid (C5), Isovaleric acid (C5), Valeric acid (C5), and Caproic acid (Hexanoic acid, C6), is determined. 
     
     
       11. A method for determining in a sample, by mass spectrometry, the presence, absence, or amount of one or more analytes selected from the group consisting of Acetic acid (C2), Propionic acid (C3), Butyric acid (C4), Isobutyric acid (C4), 2-Methyl-butyric acid (C5), Isovaleric acid (C5), Valeric acid (C5), Caproic acid (Hexanoic acid, C6), 3-Methylvaleric acid, 4-Methylvaleric acid (Isocaproic acid), and combinations thereof, wherein the run time is less than six minutes, the method comprising:
 a) subjecting the sample to chromatographic separation [prior to being subjected to an ionization source]; 
 b) subjecting the sample to an ionization source under conditions suitable to produce one or more ions detectable by mass spectrometry from each of the one or more analytes, wherein the one or more analytes are derivatized using at least 2,4-Difluorophenyl Hydrazine Hydrochloride prior to ionization; 
 c) measuring, in a single injection, by mass spectrometry, the amount of the one or more ions from each of the one or more analytes; and 
 d) using the measured amount of the one or more ions to determine the amount of each of the one or more analytes in the sample. 
 
     
     
       12. The method of  claim 11 , wherein the chromatographic separation is performed using liquid chromatography and wherein the liquid chromatography is selected from the group consisting of high-performance liquid chromatography, ultra high performance liquid chromatography, and turbulent flow liquid chromatography. 
     
     
       13. The method of  claim 11 , wherein pivalic acid interference is eliminated. 
     
     
       14. The method of  claim 11 , wherein the sample is also derivatized using 3-Nitrophenylhydrazine Hydrochloride. 
     
     
       15. The method of  claim 11 , wherein the mass spectrometer is operated in negative mode. 
     
     
       16. The method of  claim 11 , wherein the one or more ions used to determine the amount of each of the one or more analytes are one or more ions selected from the ions in Tables 4 and 5. 
     
     
       17. A kit comprising two or more isotopically labeled analogues as internal standards for each of two or more analytes selected from the group consisting of Acetic acid (C2), Propionic acid (C3), Isobutyric acid (C4), Butyric acid (C4), 2-Methyl-butyric acid (C5), Isovaleric acid (C5), Valeric acid (C5), Caproic acid (Hexanoic acid, C6), Lactic acid, Pyruvic acid, Fumaric acid, Succinic acid, Malic acid, alpha-Ketoglutaric acid, Aconitic acid, Citric acid, Isocitric acid, and combinations thereof, wherein one of the two or more isotopically labeled analogues is for an analyte selected from the group consisting of: Acetic acid (C2), Propionic acid (C3), Butyric acid (C4), Isobutyric acid (C4), 2-Methyl-butyric acid (C5), Isovaleric acid (C5), Valeric acid (C5), Caproic acid (Hexanoic acid, C6), 3-Methylvaleric acid, 4-Methylvaleric acid (Isocaproic acid); and a different one of the two or more isotopically labeled analogues is for an analyte selected from the group consisting of Lactic acid, Pyruvic acid, Fumaric acid, Succinic acid, Malic acid, alpha-Ketoglutaric acid, Aconitic acid, Citric acid, Isocitric acid, and packaging material and instructions for using the kit. 
     
     
       18. The kit of  claim 17  further comprising derivatization reagents, catalyst reagents, calibration standards, or quality control samples.